Oligonucleotides have become indispensable tools in modern molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for synthesizing oligonucleotides.
The synthesis of oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See e.g., Methods in Molecular Biology, vol. 20: Protocols for Oligonucleotides and Analogs, pp. 165-189 (S. Agrawal, Ed., Humana Press, 1993); Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., 1991); and Uhlmann and Peyman, supra. Agrawal and Iyer, Curr. Op. in Biotech, vol. 6:12, 1995; and Antisense Research and Applications (Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993). Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al., J. Molec. Biol., vol. 72:209, 1972, discloses phosphodiester chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett., vol. 34:3143-3179 (1978), discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to synthesis. Beaucage and Caruthers, Tetrahedron Lett, vol. 22:1859-1862 (1981), discloses the use of deoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawal and Zamecnik, U.S. Pat. No.5,149,798 (1992), discloses optimized synthesis of oligonucleotides by the H-phosphonate approach.
Both of these modern approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. Agrawal and Goodchild, Tetrahedron Lett., vol. 28:3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al., Biochemistry, vol. 23:3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager et at., Biochemistry, vol. 27:72 (1988), discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal et al., Proc. Ant. Acad. Sci. USA, vol. 85:7079-7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.
Synthesis of certain types of modified oligonucleotides remains problematic, however. For example, synthesis of oligonucleotides containing phosphorodithioate internucleoside linkages has been plagued by oxidation of the phosphorodithioate internucleoside linkage to a phosphorothioate internucleoside linkage. Using a .beta.-cyanothyl group to protect the sulfur moiety from oxidizing during the phosphorodithioate synthesis has been used in a previous approach with limited success and has resulted in high levels 8-9% of contaminanting phosphormonothioate incorporated into the phosphorodithioate product (Dahl et al, Acta Chem. Scand. 1989, 43, 896-901; Dahl et al, Tetrahedron Lett. 1990, 31, 3489-3492; Bjergarde et al, Nucleic Acid Res. 1991, 19, 5843-5850). Beaton et al, Oligonucleotides and Analogues: A practical approach; Eckstein, Ed.; IRL Press 1991;pp 109-135, discloses an improved procedure for synthesizing phosphorodithioate oligonucleotides. However, even in this procedure, at least 2-4% of the phosphorodithioate internucleoside linkages were oxidized to phosphorothioates and moreover, the synthons containing the 2,4-dichlorobenzyl group used to block the sulfur are highly unstable to oxidation and do not remain stable through the course of a single synthesis. Wiesler and Caruthers, J. Org. Chem., 1996, 61, 4272-4281, disclose yet another improved procedure for phosphorodithioate synthesis, however this procedure is also plagued with 2-5% phosphoromonothioate contamination incorporated into the phosphorodithioate oligonucleotide product.
One attractive feature of the phosphorodithioate internucleoside linkage is that it is achiral. Thus, this internucleoside linkage can be used to make oligunucleotides which are stereochemically pure. In contrast, the phosphorothioate internucleoside linkage exists as R.sub.p and S.sub.p enantiomers. Thus, oligonucleotides containing phosphorothioate internucleoside linkages exist as racemic mixtures which contain 2.sup.n sterichemically distinct species, wherein n represents the number of phosphorothioate internucleoside linkages present in the oligonucleotide. Accordingly, even low levels of oxidation of the phosphorodithioate internucleoside linkages to phosphorothioates can convert a stereochemically pure oligonucleotide preparation to a relatively complex racemic mixture.
There is, therefore, a need for improved reagents and processes for phosphorodithioate oligonucleotide synthesis. Ideally, such reagents and processes should be suitable for use in existing oligonucleotide synthesis protocols. There is further a need for oligonucleotides having exclusively phosphorodithioate internucleoside linkages as well as for oligonucleotides which contain phosphorothioate linkages.